This invention relates to a new method and means for detecting and assaying of various pathogens, toxins, drugs, or hormones, or their antibodies and more particularly to improvements therein.
A large number of clinical tests are based on radioimmunoassay, which is an extension of fluorescent immunoassay as well as of isotope dilution analysis. These tests include primarily the assay of a large number of hormones, primarily polypeptides, such as parathyroid or human growth hormone, as well as steroid hormones. The same methods can be extended, however, to the detection and assay of various pathogens or toxins and in fact of any drug or chemical that can be conjugated with a protein. The antibodies of these species can be assayed in a similar manner. The method is highly specific and highly sensitive and replaces bioassay methods as well as clinical analysis when very minute amounts of material have to be determined.
The methodology of radioimmunoassay has been reviewed many times and there have been recently published two volumes on the topic, one of which is Radioimmunoassay Methods, by Kirkham and Hunter, and published by Churchill Livingston, Edinburgh (1971). The other is Principles of Competitive Protein Binding Assays, by Odell and Daughaday, published by J. B. Lippincott & Co., Philadelphia (1971). In principle the radioimmunoassay comprises mixing a known amount of radioisotope labeled antigen (or antibody) with a solution carrying antibodies (or antigens), the quantity of which is desired to be determined. The labeled antigens (or antibodies) combined with the unlabeled antibodies (or antigens) in a known ratio. The labeled antigens (or antibodies) which combined with unlabeled antibodies (or antigens) are called bound, and the labeled antigens (or antibodies) which are not combined, are called unbound. The unknown concentration of the antigen (or antibody) in the sample solution is then derived from the ratio of the bound to unbound labeled tracer antigens (or antibodies). In all cases, it is necessary to separate the bound complex from the unbound labeled antigen or antibody and to determine the amount of the radioisotope in one of these forms as a function of concentration. Of the numerous methods of separating the unbound material from the bound complexes, the adsorption onto solid particles and the double antibody technique are the most popular. The radioactivity may be assayed in the supernatant or in the solid phase. Radioactivity is determined in the usual manner, using a Geiger counter, for example.
The labeling of polypeptide or conjugated protein antigens, or of antibodies, with a radioactive tracer has, however, serious limitations and drawbacks. The ultrahigh sensitivity required by this methodology (determination of subnanogram quantities of proteins) makes it imperative to use short lived isotopes at as high a specific activity as possible. It is evident, therefore, that the labeled antibodies or antigens have a rather short half-life, not only because of the limited physical half-life of the labeling radioisotope but also because of the autoradiolytic damage to the labeled protein molecules, which may readily lead to their inactivation. The practical half-life of such labeled antibodies is, therefore, only a few days, and poses a severe limitation on the whole methodology. In fact, radioimmunoassay is limited today to research laboratories capable of synthesizing the labeled antibodies or antigens, purifying the labeled protein without loss of immunological activity, and assaying it at frequent intervals to assure its antigenic activity.